Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is an established drug target of antimalarials such as pyrimethamine, which blocks the DHFR function. Basic studies on uptake of prodrugs and regulation of expression of DHFR-TS suggest that TS may also be a suitable target for malaria chemotherapy. This concept has received additional inspiration from the availability of the malaria DHFR-TS crystal structure and from vast efforts by pharmaceutical companies to develop medicinal chemistry against human TS for treatment of cancer. The central hypothesis of the present Program Project application is that folate-based TS inhibitors will be safe and effective for malaria chemotherapy when used in the presence of thymidine. The hypothesis rests on several key concepts and observations: POTENCY: Lead compounds display Ki approximately 1 nM for TS enzyme and EC50 below 100 nM against parasite cells. TARGET SPECIFICITY: As little as 10 mu M thymidine offers complete protection to mammalian cells. TARGET SPECIFICITY: In contrast, 10 mu M thymidine offers no protection to Plasmodium cells. SELECTIVITY VULNERABILITY: Malaria cells cannot overproduce DHFR-TS protein in response to antifolates. SELECTIVITY VULNERABILITY: There may be opportunities for malaria-specific TS binding. REDUCED DEVELOPMENT BARRIERS: Even without thymidine augmentation, several folate-based TS inhibitors have passed human Phase I, II, and III clinical trials for cancer treatment. The present Program Project has three specific aims: (i) To test a battery of TS inhibitors against P. falciparum for potency and emergence of resistance; (ii) To generate improved new TS inhibitors using structural and computational tools; and (iii) To measure efficacy and safety of TS inhibitors in mice and protective thymidine levels in humans. The team of established investigators offers highly complementary experiences ranging from physical biochemistry to malaria clinical trials in a U.S. hospital. Successful completion of this project will set the stage for testing malaria-specific TS inhibitors in humans and provide an exciting opportunity to take the uncommon step from basic science to the clinic with confidence.